1,720,987 research outputs found
The ICSI procedure from past to future: a systematic review of the more controversial aspects
Full text linkBackground: ICSI is currently the most commonly used assisted reproductive technology, accounting for 70-80%of the cycles performed. This extensive use, even excessive, is partly due to the high level of standardization reached by the procedure. There are, however, some aspects that deserve attention and can still be ameliorated. The aim of this systematic review was to evaluate the results of available publications dealing with the management of specific situations during ICSI in order to support embryologists in trying to offer the best laboratory individualized treatment. Methods: This systematic review is based on material obtained by searching PUBMED between January 1996 and March 2015. We included peer-reviewed, English-language journal articles that have evaluated ICSI outcomes in the case of (i) immature oocytes, (ii) oocyte degeneration, (iii) timing of the various phases, (iv) polar body position during injection, (v) zona-free oocytes, (vi) fertilization deficiency, (vii) round-headed sperm, (viii) immotile sperm and (ix) semen samples with high DNA fragmentation. Results: More than 1770 articles were obtained, from which only 90 were specifically related to the issues developed for female gametes and 55 for the issues developed for male gametes. The studies selected for this review were organized in order to provide a guide to overcome roadblocks. According to these studies, the injection of rescue metaphase I oocytes should be discouraged due to poor clinical outcomes and a high aneuploidy rates; laser-assisted ICSI represents an efficient method to solve the high oocyte degeneration rate; the optimal ICSI timing and the best polar body position during the injection have not been clarified; injected zona-free oocytes, if handled carefully, can develop up to blastocyst stage and implant; efficient options can be offered to patients who suffered fertilization failure in previous conventional ICSI cycles. Most controversial and inconclusive are data on the best method to select a viable spermatozoa when only immotile spermatozoa are available for ICSI and, to date, there is no reliable approach to completely filter out spermatozoa with fragmented DNA from an ejaculate. However, most of the studies do not report essential clinical outcomes, such as live birth, miscarriage and fetal abnormality rate, which are essential to establish the safety of a procedure. Conclusions: This review provides the current knowledge on some controversial technical aspects of the ICSI procedures in order to improve its efficacy in specific contexts. Notwithstanding that embryologists might benefit from the approaches presented herein in order to improve ICSI outcomes, this area of expertise still demands a greater number of well-designed studies, especially in order to solve open issues about the safety of these procedures
Impaired spermatogenesis in the twitcher mouse: A morphological evaluation from the seminiferous tubules to epididymal transit
No full textSpermatogenesis is a complex process of proliferation and differentiation during male germ cell development whereby undifferentiated spermatogonial germ cells evolve into maturing spermatozoa. In this developmental process the interactions between different cell types are finely regulated, hence any disruption in these relationships leads to male infertility. The twitcher mouse, the murine model of Krabbe disease, is characterized by deficiency of galactosylceramidase, an enzyme also involved in the metabolism of the galactosyl-alkyl-acyl-glycerol, the precursor of sulfogalactosyl-alkyl-acyl-glycerol, the most abundant glycolipid in spermatozoa. Twitcher mice are sterile due to alterations of spermatogenesis resulting in the production of spermatozoa with abnormally swollen acrosomes and bent flagella, mainly at the midpiece–principal piece junction. The current study employs light, fluorescence, and electron microscopy to examine the defective spermiogenesis leading to the morphological abnormalities of mature sperm. This study reveals that alterations in germ cell development can be initially detected at the stage VIII and IX of spermatogenesis. The disrupted spermatogenetic process leads to a reduced number of elongating spermatids and spermatozoa in these mutant animals. Electron microscopy analysis demonstrates major acrosomal and chromatin condensation defects in the mutants. In addition, in twitcher mice, the epididymal architecture is impaired, with stereocilia of caput and corpus broken, detached and completely spread out into the lumen. These findings indicate that seminolipid expression is crucial for proper development of spermatocytes and spermatids and for their normal differentiation into mature spermatozoa. Abbreviations: GALC: galactosylceramidase; GalAAG: galactosyl-alkyl-acyl-glycerol; SGalAAG: sulfogalactosylalkylacylglycerol; PND: postnatal day; PAS: periodic acid-Schiff stain; TEM: transmission electron microscopy; SEM: scanning electron microscopy; PFA: paraformaldheyd
3D model of the maternal-fetal interface: challenges, recent advances and beyond
Full text linkEmbryo implantation is a complex and highly coordinated process that involves an intricate network of factors establishing intimate contact at the maternal-fetal interface. Knowledge of the human implantation process is compromised by both ethical issues, which do not allow the study of this process in vivo, and by the accuracy and reproducibility of in vitro models of human endometrium. Effective and reliable embryo implantation models are, therefore, necessary to mimic the molecular event cascade that occurs in vivo. 3D models are considered a new step to foster precision medicine and an advanced tool for the study of endometrial biology, endometrium associated diseases and to understand the complex mechanisms surrounding endometrium-embryo crosstalk. In this review we explore the various methods by which 3D cultures of endometrium and trophoblast can be created, exploring targets and applications of these in vitro models
Single nucleotide polymorphisms of USP26 in azoospermic men
No full textSome studies have focused on the association between male infertility and single nucleotide polymorphisms (SNPs) in the ubiquitin-specific protease 26 (USP26) gene, but the results are controversial. In this case-control study including both normozoospermic men and patients with nonobstructive azoospermia, we analyzed both the entire coding region and 5’ and 3’ untranslated regions of USP26 in order to identify genetic variants in this gene to investigate the role of USP26 on spermatogenesis. We reported variations in the USP26 gene sequence in 82% of azoospermic and in 50% normospermic men. The synonymous variation c.576G>A has a frequency significantly different in the azoospermic (60.2%) and normozoospermic (23.6%) groups, while the frequencies in the two groups of both c.1090C>T and c.1737G>A missense mutations did not reach statistical significance. A cluster mutation (c.371insACA, c.494T>C) was detected in 2 normozoospermic men (2.7%). In the 5’UTR we identified the -33C>T variation both in azoospermic (3.8%) and in normozoospermic (2.7%) men. In a normozoospermic man we detected the nonsense mutation c.882C>A, never reported to date. According to our results, we suggest that only the variation c.576G>A has a frequency significantly different in azoospermic compared to normozoospermic men. Moreover, the identification in a normozoospermic man of a nonsense mutation (c.882C>A) which causes the production of a truncated protein, suggests a marginal role of USP26 in male spermatogenesis. Additional studies may be useful as we cannot exclude that the other SNPs may represent risk factors for male fertility acting by an oligogenic/polygenic mechanism
Successful multiple pregnancy achieved after transfer of frozen embryos obtained via intracytoplasmic sperm injection with testicular sperm from an AZFc-deleted man
No full textObjective: To describe a case of successful triplet pregnancy after testicular sperm extraction (TESE) from a man with AZFc deletion and intracytoplasmic sperm injection (ICSI). Design: Case report. Setting: University hospital. Patient(s): A 38-year-old man affected by complete AZFc deletion and azoospermia. Intervention(s): Spermiogram, Y-chromosome microdeletion screening, TESE for sperm recovery from testicular tissue on the same day as ICSI, transfer of frozen-thawed embryos, vaginal ultrasound examination. Main Outcome Measure(s): The Y chromosome genetic status of an azoospermic patient who underwent TESE and ICSI, the fertilization and pregnancy outcome. Result(s): The patient was found to be azoospermic, and the deletion screening showed complete AZFc deletion. After TESE, the recovered testicular sperm were selected for ICSI. Three good quality embryos were obtained and were frozen due to ovarian hyperstimulation syndrome in the female partner. After transfer of the thawed embryos, a triplet pregnancy was diagnosed by vaginal ultrasonography at the seventh week of gestation. Two male and one female healthy babies were born. Conclusion(s): This is the first report of a successful triplet pregnancy after the transfer of frozen-thawed embryos in a couple in whom the male partner was azoospermic and a carrier of complete AZFc deletion. This deletion should not adversely affect a man's TESE retrieval prognosis or the fertilization, cleavage, and implantation of embryos. The offspring were healthy, although the two sons inherited the AZFc deletion. Copyright © 2010 American Society for Reproductive Medicine, Published by Elsevier Inc
Suppression of galactocerebrosidase premature termination codon and rescue of galactocerebrosidase activity in twitcher cells
No full textKrabbe's disease (KD) is a degenerative lysosomal storage disease resulting from deficiency of β-galactocerebrosidase activity. Over 100 mutations are known to cause the disease, and these usually occur in compound heterozygote patterns. In affected patients, nonsense mutations leading to a nonfunctional enzyme are often found associated with other mutations. The twitcher mouse is a naturally occurring model of KD, containing in β-galactocerebrosidase a premature stop codon, W339X. Recent studies have shown that selected compounds may induce the ribosomal bypass of premature stop codons without affecting the normal termination codons. The rescue of β-galactocerebrosidase activity induced by treatment with premature termination codon (PTC) 124, a well-characterized compound known to induce ribosomal read-through, was investigated on oligodendrocytes prepared from twitcher mice and on human fibroblasts from patients bearing nonsense mutations. The effectiveness of the nonsense-mediated mRNA decay (NMD) inhibitor 1 (NMDI1), a newly identified inhibitor of NMD, was also tested. Incubation of these cell lines with PTC124 and NMDI1 increased the levels of mRNA and rescued galactocerebrosidase enzymatic activity in a dose-dependent manner. The low but sustained expression of β-galactocerebrosidase in oligodendrocytes was sufficient to improve the morphology of the differentiated cells. Our in vitro approach provides the basis for further investigation of ribosomal read-through as an alternative therapeutic strategy to ameliorate the quality of life in selected KD patients. © 2016 Wiley Periodicals, Inc
Assessment of sperm chromosomal abnormalities using fluorescence in situ hybridization (FISH): implications for reproductive potential
Full text linkPurpose: Chromosomal abnormalities play an important role in male infertility, which is becoming a significant issue in human fertility. Aim of this study was to evaluate the incidence of spermatic aneuploidies and diploidies in human sperm, according to semen parameters. Methods: We performed semen analysis according to the 6th edition of WHO criteria in 50 male subjects; samples were divided into normozoospermic (n = 23) or those with altered seminal parameters (n = 27). To assess chromosomal numerical alterations of sperm, fluorescence in situ hybridization (FISH) was used. Result: A significant increase in aneuploidies and diploidies was observed in samples with altered seminal parameters. Furthermore, stratifying this group, we observed a significant increase in aneuploidies and total abnormalities in oligozoospermic, asthenoteratozoospermic (AT), and oligoteratoasthenozoospermic (OAT) samples compared to normozoospermic. Conclusion: Our results showed the correlation between altered seminal parameters and numerical chromosomal abnormalities, confirming that sperm FISH analysis could be an additional clinical tool to assess reproductive potential in infertile males. Moreover, our results point to the importance of updating the normality ranges for detecting chromosomal aneuploidies using FISH
Proteostasis network alteration in lysosomal storage disorders: Insights from the mouse model of Krabbe disease
No full textIn Krabbe disease, a mutation in GALC gene causes widespread demyelination determining cell death by apoptosis, mainly in oligodendrocytes and Schwann cells. Less is known on the molecular mechanisms induced by this deficiency. Here, we report an impairment in protein synthesis and degradation and in proteasomal clearance with a potential accumulation of the misfolded proteins and induction of the endoplasmic reticulum stress in the brain of 6-day-old twitcher mice (TM) (model of Krabbe disease). In particular, an imbalance of the immunoproteasome function was highlighted,useful for shaping adaptive immune response by neurological cells. Moreover, our data show an involvement of cytoskeleton remodeling in Krabbe pathogenesis, with a lamin meshwork disaggregation in twitcher oligodendrocytes in 6-day-old TM. This study provides interesting protein targets and mechanistic insight on the early onset of Krabbe disease that may be promising options to be tested in combination with currently available therapies to rescue Krabbe phenotype
Quantification of psychosine in the serum of twitcher mouse by LC-ESI-tandem-MS analysis
No full textGloboid cell leukodystrophy or Krabbe disease is an inherited autosomal recessive disorder caused by
mutations in the galactosylceramidase (GALC) gene. Deficiency of GALC results in the accumulation of
a highly cytotoxic metabolite galactosylsphingosine (psychosine). In the present study, we describe the
development and validation of a sensitive and specific LC–ESI-tandem-MS method for the determination
of psychosine in the serum of twitcher mice, the naturally occurring animal model of this disease. The
method was validated in terms of accuracy, precision, specificity, linearity and sensitivity. Calibration
plots were linear over the concentration range of 2.5–50 ng/mL. Recovery of psychosine from serum was
in the range 94.20–98.02%. The results of this study show that in the affected mice the concentration of
psychosine (ranging from 2.53 to 33.27 ng/mL) increased significantly with the progression of the disease.
The maximum level (33.27 ng/mL) was detected in the serum of one of the twitcher mice sacrificed
at 40 PND. Psychosine was not detected at significant levels in wild type mice. These results clearly
demonstrate that this noninvasive, rapid, and highly sensitive LC–ESI-tandem-MS method is a very useful
approach for the detection of psychosine in serum. © 2013 Elsevier B.V
Expression of microtubule associate protein 2 and synaptophysin in endometrium: high levels in deep infiltrating endometriosis lesions
No full textObjective To assess whether healthy endometrium, eutopic endometrium, and endometriotic lesions express nerve growth factor (NGF), microtubule-associated protein 2 (MAP-2), and synaptophysin (SYP). Design Molecular study in tissue extracts. Setting University hospital. Patient(s) A group of women (n = 70), divided as [1] healthy controls (n = 30) and [2] with endometriosis (n = 40), was included. Intervention(s) From the healthy control group an endometrial specimen was collected by hysteroscopy (proliferative phase, n = 16; secretive phase, n = 14). Endometriotic and endometrial specimens were collected from women undergoing laparoscopic surgery for endometriosis, endometrioma (OMA) (n = 20), or deep infiltrating endometriosis (DIE) (n = 20). Main Outcome Measure(s) To assess expression of NGF, MAP-2, and SYP messenger RNA (mRNA) levels in endometrium and in endometriosis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and protein localization by immunofluorescence. Cultures of human endometrial stromal cells were used to evaluate the effect of tumor necrosis factor (TNF)-α on NGF and SYP. Result(s) Endometrial tissue from control expressed mRNA for NGF, MAP-2, and SYP, without any difference between proliferative and secretive phase. The DIE and OMA lesions showed the highest NGF mRNA expression, significantly higher than in eutopic endometrium and control. In DIE lesions SYP mRNA expression was higher than in OMA or in eutopic endometrium or controls. Immunofluorescence analysis of NGF, MAP-2, and SYP showed a slightly more intense positive signal in endometriotic lesions. Exposure to TNF-α increased NGF and SYP mRNA expression in endometrial culture cells. Conclusion(s) The present study revealed the presence of two selected neuronal markers, MAP-2 and SYP mRNAs and protein expression, in eutopic endometrium and in endometriotic lesions
- …
